Chemical Composition and Antimicrobial Activities of Essential Oils of Some Coniferous Plants Cultivated in Egypt.

Family Cupressaceae is the largest coniferous plant family. Essential oils of many species belonging to family Cupressaceae are known to have several biological activities specially antimicrobial activity. The essential oils from aerial parts of Calocedrus decurrens Torr., Cupressus sempervirens stricta L. and Tetraclinis articulata (Vahl) Mast. were prepared by hydrodistillation. The chemical composition of the essential oils has been elucidated by gas chromatography-mass spectroscopy analysis. The prepared essential oils were examined against selected species of Gram-positive, Gram-negative bacteria and Candida species. Broth dilution methods were used to detect minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC). Sixteen compounds were identified in the essential oils of both Calocedrus decurrens and Cupressus sempervirens L. and fifteen compounds were identified in the essential oil of Tetraclinis articulata. δ-3-Carene (43.10%), (+)-Cedrol (74.03%) and Camphor (21.23%) were the major constituents in the essential oils of Calocedrus decurrens, Cupressus sempervirens L. and Tetraclinis articulata, respectively. The essential oils showed strong antimicrobial activities against the selected microorganisms in concentration range 0.02 3- 3.03 µL/mL. This study could contribute to the chemotaxonomic characterization of family Cupressaceae. In addition, it proved that the essential oils under investigation possess potential antimicrobial properties.


Introduction
The use of essential oils to control many diseases and their effective usage as antimicrobial agents (1)(2)(3)(4)(5) in addition to their use as functional ingredients in foods, drinks, toiletries and cosmetics is gaining momentum, both for the growing interest of consumers in ingredients from natural sources and also because of increasing concern about potentially harmful synthetic additives (6). Essential oils are complex mixture of natural compounds, mostly of plant origin, extremely volatile and with an intense odour. Even if they represent only a small fraction of the plant from which they are derived, they give the whole plant the characteristic of aromatic smell for which these plants are employed by drug, food and perfume industries (7). The species that show the largest (31.31%), δ-3-carene (21%) and α-pinene (9.2%) while Robert & Sanko (22) proved that the oils of C. decurrens from two populations in Oregon and one disjunct population in southern California were high in δ-3 carene (15.20%-20.00%), limonene (18.21-23.62%), α-pinene (8.7-15.8%), terpinolene (5.72-8.01%), α-fenchyl acetate (3,5-9.71%) and cedrol (0.8-1.2%) (21,22).
The main objective of this study was to identify the chemical composition of the essential oils from the aerial parts of C. decurrens, C. sempervirens and T. articulata cultivated in Egypt, and to determine their antimicrobial activities, in an attempt to contribute to the use of these essential oils as alternative products.

Preparation of the essential oil
The essential oils were prepared from the fresh aerial parts of C. decurrens, C. sempervirens and T. articulata (500 g) by hydro distillation using Clevenger-type apparatus. The oils were dried over anhydrous sodium sulphate and stored in sealed glass vials at 4-6 o C prior to analysis. Percentage yields were determined according to Egyptian Pharmacopoeia 1984 (32). Percentage yields and physical properties of the essential oils are illustrated in Table 1.

GC/ MS analysis of the essential oil
The prepared essential oils were subjected to GC/MS analysis using Shimadzu GC//MS -QP 5050 A, fitted with a DB-1 fused silica capillary column (30 m, 0.53 mm ID, 1.51 µM film thickness). Software Class 5000. searched library: Wiley 229 LIB. Carrier gas: Helium (flow rate 1 mL/min.). Ionization mode: EL (70 ev). Temperature program: 40 0 C (static for 2 min) then gradually increasing at a rate of 2 0 C/ min up to 250 0 C (static for 7.50 min). Detector temperature 250 0 C. Injector temperature 250 0 C.

Identification of the essential oil
Compounds were identified by comparison of their retention indices (RI), obtained on a non-polar DB-1 column relative to C5-C24 n-alkanes, with those provided in the literature, in addition to Library searched data base Willey 229LIB and by comparing mass fragmentation patterns with those of the available references and with published data (33-35). The percentage composition of the essential oils was determined by computerized peak area measurements. Results were calculated as mean values after two injections for each essential oil. Results are presented in Tables 2,3. Testing for antimicrobial activities Minimum Inhibitory Concentrations (MICs) were determined using the broth microdilution method (36, 37). Determination of antimicrobial activities against yeast was achieved by microdilution method using serial dilutions of the essential oils (0.008-64.00 μL/mL), which were prepared in 96-well microtiter plates by microdilution method using RPMI-1640 media (Sigma, St. Louis, MO, USA) buffered with MOPS (Sigma). The antibacterial activities were determined by serial dilutions of the essential oils (0.03-128.00 μL/mL, DMSO) in Mueller-Hinton broth (Merck, Darmstadt, Germany). Test yeasts or bacteria strains were suspended in media and the cell densities were adjusted to . In addition, positive (medium with inoculums but without essential oil) and negative (Uninoculated medium, 200 μL) growth controls were prepared. The growth in each well was compared with the growth in the control well. MICs were visually determined and defined as the lowest concentration of the essential oil produced ≥ 50% growth inhibition for fungi and ≥95% growth reduction for bacteria compared with the growth in the control well. Each experiment was performed in triplicate. Gentamycin (Sigma-Aldrich, Steinheim, Germany) and nystatin (Merck, Darmstadt, Germany) in concentration range (0.001-64.00 µg/mL, sterile distilled water) were used as standard antibacterial and antifungal drugs, respectively. In addition, media from wells with fungi showing no visible growth were further cultured on Sabouraud dextrose agar (Merck, Darmstadt, Germany) and from wells with bacteria showing no visible growth on Mueller-Hinton agar (Merck, Darmstadt, Germany) to determine the minimum fungicidal concentrations (MFC) and minimum bactericidal concentrations (MBC).

Statistical Analysis
Results were expressed as the mean ± standard deviation; statistical analysis of experimental results were based on the analysis of variance method. Differences were considered statistically significant at the level of P < 0.001.

Results and Discussion
A noticeable variation was observed in the percentage yield of hydrodistilled essential oil prepared from the aerial parts of calocedrus incense cedar, C. sempervirens, and T. articulata, cultivated in Egypt yielding (1.41%, 0.32% and 1.71%), respectively. Results of GC/MS analysis of essential oils of plants under investigation showed qualitative and quantitative variations. Sixteen compounds were determined in essential oils of C. decurrens and C. sempervirens, while fifteen compounds were identified in essential oil of T. articulata.

Mean (µL/mL) ± Standard Deviation Gentamycin Mean (µg/mL) ± Standard Deviation
The antibacterial activities of C. decurrens, C. sempervirens and T. articulata essential oils against the tested Gram-positive and Gramnegative baeteria are shown in Table 4. The essential oils under investigation inhibited the growth of S. epidermidis at concentrations 0.023-0.442 µL/mL. Essential oil of C. sempervirens showed inhibition of the growth of S. pyogenes at concentration 0.84 µL/mL, while essential oils of C. decurrens and T. articulata at concentrations up to 64 µL/mL showed no inhibition of the growth of S. pyogenes. E. coli showed no susceptibility to essential oil of C. decurrens, while all the tested Gram-negative microorganisms showed growth inhibition by the effect of essential oils of C. sempervirens and T. articulata at concentrations range 0.037-3.031 µL/mL. In addition, all the tested essential oils excreted bactericidal activities against all the susceptible Gram-positive and gramnegative microorganisms at concentration range 0.155-6.72 µL/mL. C. sempervirens showed the highest antibacterial activities against most of the tested bacterial strains.
The antifungal activities of the essential oils of C. decurrens, C. sempervirens and T. articulata against tested yeast strains are shown in Table 5. Essential oil of C. decurrens showed no activities against all the tested yeast strains except C. parapsilosis which showed growth inhibition at concentration 0.824 µL/ mL. C. glabrata and C. krusei showed no susceptibility to any of the studied essential oils, while essential oils of C. sempervirens and T. articulata inhibited the growth of C. albicans and C. parapsilosis at concentration range 0.42-0.757 µL/mL. The tested essential oils showed MFC against the susceptible Candida species ranging from 1.148 µL/mL to 2.828 µL/mL. C. sempervirens essential oil showed the highest fungicidal activities followed by T. articulata and C. decurrens.
From this study it could be concluded that the essential oils under investigation possess antimicrobial activities. C. sempervirens essential oil has the most potential antimicrobial properties followed by T. articulata essential oil. The results of the study are inaccordance with the previous investigations of essential oil of T. articulata, which proved the presence of α-pinene, camphor, linalool acetate, caryophyllene, alloaromadendrene, bornyl acetate and limonene as the major constituents in several studies of essential oil of different organs of T. articulata in different countries (27-31, 38, 39). Previous investigations on essential oil of C. decurrens from USA and Taiwan proved the presence of α-pinene, δ-3-carene, terpinene, terpinolene, linalool, α-fenchyl acetate, β-caryophyllene and cedrol (21, 22)] which were detected in this study. While, a previous study on essential oil of leaves of C. sempervirens cultivated in Egypt proved that cedrol constituted the major constituent of the oil followed by δ-3-carene and α-pinene (18) whilst the essential oil of the leaves of the plant cultivated in Tunisia showed the presence of α-pinene as a major component followed by δ-3carene and limonene (19). As the composition of the essential oils revealed intraspecific chemical variability among the same species growing in different localities and different environmental conditions, this study could contribute to the chemotaxonomic characterization of family Cupressaceae. From this study, it was concluded that the essential oils of plants of family Cupressaceae which were under investigation in this study showed low presence of non-oxygenated sesquiterpenes ranging from 0.93% to 5.26%. In addition to the occurrence of variable percentages of non-oxygenated monoterpenes (9.28%-55.4%), oxygenated monoterpenes (7.38%-83.70%) and oxygenated sesquiterpenes (1.47%-80.90%). Meanwhile, α-pinene is the only common compound that was detected in all the tested essential oils.
The significant antimicrobial effect could be attributed to the presence of high percentage of oxygenated compounds specially cedrol (40). The results of antimicrobial activities proved in this study are in agreement with previous studies on the antimicrobial activities of essential oils of the plants under investigation (19, 27). Essential oil of the leaves of Tetraclinis articulata from Algeria showed antifungal activities against Fusarium species (27). Moreover, the essential oil of Cupressus sempervirens from Tunisia inhibited the growth of bacteria, fungi and yeast (19).

Conclusion
We believe that the present investigation together with previous studies provide support to the antimicrobial properties of the tested essential oils. They could be used as antimicrobial supplement in the developing countries towards the development of new therapeutic agents. Additional in-vivo studies and clinical trials would be needed to justify and further evaluate the potential of these oils as antimicrobial agents.